U.S. patent application number 10/549176 was filed with the patent office on 2006-12-07 for angiotensin-converting enzyme inhibitory peptides.
Invention is credited to Sumio Asami, Taeko Iino, Kyoichi Ogura.
Application Number | 20060276627 10/549176 |
Document ID | / |
Family ID | 33027809 |
Filed Date | 2006-12-07 |
United States Patent
Application |
20060276627 |
Kind Code |
A1 |
Ogura; Kyoichi ; et
al. |
December 7, 2006 |
Angiotensin-converting enzyme inhibitory peptides
Abstract
It is intended to provide ACE inhibitory tripeptides which are
not easily digested by digestive enzymes after being orally taken
and thus have fewer tendencies to lose their ACE inhibitory
activity in vivo. More specifically, 3 tripeptides having an ACE
inhibitory activity and showing a hypotensive effect in an animal
experiment are discovered from a thermolysin digestion product of
sesame. These tripeptides respectively have amino acid sequences
Leu-Ser-Ala, Val-Ile-Tyr and Leu-Val-Tyr and show an angiotensin
converting enzyme inhibitory activity.
Inventors: |
Ogura; Kyoichi; (Kyoto-shi,
Kyoto, JP) ; Iino; Taeko; (Shiga, JP) ; Asami;
Sumio; (Osaka, JP) |
Correspondence
Address: |
DRINKER BIDDLE & REATH (DC)
1500 K STREET, N.W.
SUITE 1100
WASHINGTON
DC
20005-1209
US
|
Family ID: |
33027809 |
Appl. No.: |
10/549176 |
Filed: |
March 17, 2004 |
PCT Filed: |
March 17, 2004 |
PCT NO: |
PCT/JP04/03588 |
371 Date: |
August 14, 2006 |
Current U.S.
Class: |
530/331 ;
424/439 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 9/12 20180101; A61K 38/00 20130101; C07K 5/0808 20130101 |
Class at
Publication: |
530/331 ;
514/018; 424/439 |
International
Class: |
A61K 38/05 20060101
A61K038/05; C07K 5/06 20060101 C07K005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 18, 2003 |
JP |
2003074488 |
Claims
1. A tripeptide having an amino aid sequence selected from the
group consisting of Leu-Ser-Ala, Val-Ile-Tyr and Leu-Val-Tyr.
2. A tripeptide according to claim 1 which is obtained by digesting
a protein originating from one or more materials selected from
among rice, wheat, barley, oat, corn, kidney bean, broad bean,
soybean, mung bean and sesame with thermolysin.
3. An edible composition containing one or more added tripeptides
having an amino aid sequence selected from the group consisting of
Leu-Ser-Ala, Val-Ile-Tyr and Leu-Val-Tyr.
4. An angiotensin converting enzyme inhibitor containing, as the
active ingredient, one or more tripeptides having an amino aid
sequence selected from the group consisting of Leu-Ser-Ala,
Val-Ile-Tyr and Leu-Val-Tyr.
5. A hypotensive agent containing, as the active ingredient, one or
more tripeptides having an amino aid sequence selected from the
group consisting of Leu-Ser-Ala, Val-Ile-Tyr and Leu-Val-Tyr.
6. An edible composition according to claim 3 comprising from 0.001
mg to 100 mg of said added tripeptides in a single intake dose.
7. An angiotensin converting enzyme inhibitor according to claim 4
comprising from 0.001 mg to 100 mg of said tripeptide in a single
dose for oral administration.
8. A hypotensive agent according to claim 5 comprising from 0.001
mg to 100 mg of said tripeptide in a single dose for oral
administration.
Description
FIELD OF THE INVENTION
[0001] This invention relates to peptides which inhibit angiotensin
converting enzyme and which are therefore useful as ingredients of
health foods, drugs, etc. having a hypotensive effect.
PRIOR ART
[0002] The number of patients with hypertension, which is a typical
example of life-style related diseases, is increasing year by year.
It is known that hypertension induces various complications such as
cerebral hemorrhage, subarachnoid hemorrhage, cerebral infarction,
myocardial infarction, angina, nephrosclerosis and so on. Thus,
various studies have been made on the onset mechanism of
hypertension.
[0003] As blood pressure-regulatory systems, the renin-angiotensin
system relating to the elevation of blood pressure and the
kallikrein-kinin system relating to the reduction of blood pressure
play important roles. In the renin-angiotensin system,
angiotensinogen secreted from the liver is converted into
angiotensin I by renin produced in the kidney. Angiotensin I is
further converted into angiotensin II by angiotensin converting
enzyme (ACE). Angiotensin II induces contraction of smooth vascular
muscles and thus elevates blood pressure. On the other hand,
kallikrein in the hypotensive system acts on kininogen and thus
produces bradykinin. Bradykinin has a vasodilating effect and
lowers blood pressure. However, ACE has an effect of degrading
bradykinin. That is to say, it is known that ACE participates in
the elevation of blood pressure through the above-described two
effects, i.e., producing angiotensin II which is a vasopressor
peptide and inactivating bradykinin which is a hypotensive peptide.
Therefore, it will be possible to reduce elevation of blood
pressure by suppressing the enzyme activity of ACE. Proline
derivatives such as captopril and enalapril developed as ACE
inhibitors have been widely employed in treating hypertension.
[0004] In recent years, it has been reported that peptides obtained
by digesting food materials with enzymes have an ACE inhibitory
activity. For example, there have been reported a large number of
such digestion products, e.g., a collagenase digestion product of
gelatin (Japanese Patent Public Disclosure SHO 52-148631), a
trypsin digestion product of casein (Japanese Patent Public
Disclosure SHO 58-109425, Japanese Patent Public Disclosure SHO
59-44323, Japanese Patent Public Disclosure SHO 60-23086, Japanese
Patent Public Disclosure SHO 60-23087, Japanese Patent Public
Disclosure SHO 61-36226 and Japanese Patent Public Disclosure SHO
61-36227), a thermolysin digestion product of .gamma.-zein
(Japanese Patent Public Disclosure SHO 2-32127), a pepsin digestion
product of sardine muscle (Japanese Patent Public Disclosure HEI
3-11097), a thermolysin digestion product of dried bonito (Japanese
Patent Public Disclosure HEI 4-144696), a thermolysin digestion
product of sesame protein (Japanese Patent Public Disclosure HEI
8-231588), a pepsin digestion product of .kappa.-casein (Japanese
Patent Public Disclosure 8-269088) and so on.
[0005] These ACE inhibitory peptides, being of food origin, have
significant advantages, i.e., they pose few problems of safety
(i.e., side effects, toxicity, etc.) and are edible like common
foods. However, it has been reported that the above-described
peptide products mainly comprise peptides of 5 or more amino acids
(Japanese Patent Public Disclosure SHO 52-148631, Japanese Patent
Public Disclosure SHO 58-109425, Japanese Patent Public Disclosure
SHO 59-44323, Japanese Patent Publication SHO 60-23086, Japanese
Patent Public Disclosure SHO 61-36226, Japanese Patent Public
Disclosure SHO 61-36227, Japanese Patent Public Disclosure HEI
3-11097, Japanese Patent No. 3135812 and Japanese Patent Public
Disclosure HEI 8-269088). It has been pointed out that peptides
consisted of longer amino acid chains cannot achieve a hypotensive
effect of the level expected based on the strong ACE inhibitory
activity in vitro, probably because they are susceptible to
digestion by digestive enzymes in the body and thus lose the ACE
inhibitory activity, or, even if they remain not digested, they are
not easily absorbed because of their bulky molecular
structures.
SUMMARY OF THE INVENTION
[0006] Accordingly, an object of the present invention is to
provide ACE inhibitory tripeptides which are not easily digested by
digestive enzymes when taken orally and thus have fewer tendencies
to lose their ACE inhibitory activity in vivo.
[0007] In the present invention, it is also intended to provide
edible (food/drink) compositions, angiotensin converting enzyme
inhibitors and hypotensive agents comprising one or more of the
above-described tripeptides.
BRIEF DESCRIPTION OF THE DRAWING
[0008] FIG. 1 is a graph showing the results of an examination on
the hypotensive effects of the peptides according to the present
invention with the use of spontaneous hypotensive rats.
DETAILED DESCRIPTION OF THE INVENTION
[0009] The present inventors searched for ACE inhibitory peptides
consisted of not more than 3 amino acids, assuming that thermolysin
digestion products of food materials would contain peptides capable
of overcoming the above-described problems. As a result, they
succeeded in discovering 3 tripeptides in a thermolysin digestion
product of sesame, said tripeptides having an ACE inhibitory
activity and showing a hypotensive effect in an animal experiment.
The present invention was accomplished based on these findings.
[0010] Accordingly, the present invention provides tripeptides
respectively having the amino acid sequences Leu-Ser-Ala,
Val-Ile-Tyr and Leu-Val-Tyr and showing an angiotensin converting
enzyme inhibitory activity.
[0011] The present invention further provides edible compositions,
angiotensin converting enzyme inhibitors and hypotensive agents
containing one or more of the above-described tripeptides.
[0012] The tripeptides according to the present invention may be
produced by chemical synthesis. However, in an embodiment of the
invention wherein the tripeptides are added to foods, drinks or
oral drugs to exploit their ACE inhibitory activity, it is
preferred to produce an edible composition enriched with at least
one of the above-described 3 tripeptides by digesting vegetable
protein originating in sesame or the like with thermolysin and
further purifying the same.
[0013] As a vegetable protein source, use can be made of
protein-rich plant tissues (preferably seeds), for example, cereals
such as rice, wheat, barely, oat and corn, or beans such as kidney
bean, broad bean, soybean and mung bean and sesame.
[0014] When the peptides according to the invention are to be
obtained by digestion with thermolysin, the treatment procedure
varies depending on the properties of the starting material. It is
preferred that, as a pretreatment, the material is first defatted
by, for example, removing the juice by squeezing or extracting the
fat with a solvent such as an alcohol, acetone, hexane, etc. To
improve the efficiency of the digestion of the starting material
with thermolysin, it is also preferred that the starting material
be finely milled and then suspended in water under stirring. In the
case of a hardly soluble protein, it is also possible to employ
another pretreatment such as addition of sodium hydroxide or
heating to thereby uniformly dissolve or suspend the protein. Then
thermolysin is added thereto in an appropriate amount, preferably
from 500 to 50000 PU per g of the protein and the protein digestion
reaction is carried out at pH 5 to 9, at a temperature of 10 to
80.degree. C., for 0.5 to 48 hours either in a stationary state or
under stirring. ("PU" means "protease unit" and 1 PU is defined as
the amount of an enzyme giving an increase in non-protein Folin's
color equivalent to 1 .mu.g of tyrosine per min using milk casein
as the substrate at pH 7.2 and at 35.degree. C.) To examine whether
or not sufficient progress of the reaction has been made (i.e., the
reaction is sufficient for obtaining the purposed tripeptides), use
can be made of a method comprising "applying the liquid reaction
mixture to high-performance liquid chromatography using an ODS
column and determining the elution pattern by measuring the
absorbance at 210 nm". The reaction is ceased by adding, for
example, hydrochloric acid. Alternatively, the thermolysin may be
inactivated by heating. It is also possible to cease the reaction
by both the adding hydrochloric acid and heating. The liquid
reaction mixture is subjected to centrifugation, filtration, etc.
and the precipitate is removed. The filtrate thus obtained is
neutralized with sodium hydroxide or hydrochloric acid and then
concentrated. Further, off-flavor (for example, bitterness,
harshness, offensive odor, etc.) can be removed if necessary by
treating it with activated charcoal. The sesame peptides thus
obtained contain Leu-Ser-Ala, Val-Ile-Tyr and Leu-Val-Tyr each in
an amount of from 0.001% by weight to 0.1% by weight.
[0015] The thermolysin digestion product obtained in the
above-described manner can be used as the tripeptide composition of
the invention with or without a further treatment with an ion
exchange resin, a high-porous polymer resin, etc. to remove
high-molecular weight proteins, to thereby provide a partially
purified product rich in the tripeptides of the present invention.
These digestion product and partially purified product in general
will be sometimes referred to hereinafter as "tripeptide-rich
composition". Such a composition may be further treated, if
necessary, by activated charcoal to remove off-flavor (for example,
bitterness, harshness, offensive smell, etc.) before using.
[0016] To obtain a purified preparation of the peptides of the
invention, the above-described concentrate is subjected to gel
filtration column chromatography, chromatography with the use of an
ion exchange resin or a high-porous polymer resin, affinity
chromatography, etc. and peptide fractions of the invention having
the ACE inhibitory activity are combined. Next, the combined active
fractions can be purified by a method commonly employed in
purifying peptides, for example, high-performance liquid
chromatography with the use of a reversed phase column such as an
ODS column or a C30 column to thereby provide single forms of the
peptides in a substantially pure state. The tripeptides of the
invention can be obtained not only from sesame (e.g., Sesamum
indicum L.) but also from cereals such as rice (e.g., Oryza sativa
L.), wheat (e.g., Triticum aestivum L., T. durum Desf., T. turgidum
L., T. pyramidale (Delile) Perciv. non Delile ex Schult, T.
abyssinicum Vavilov, and T. carthlicum Nevski), barley (e.g.,
Hordeum vulgare L.), oat (e.g., Avena sativa L.) or corn (e.g., Zea
mays L.), or beans such as kidney bean (e.g., Phaseolus vulgaris
L.), broad bean (e.g., Vicia faba L.), soybean (e.g., Glycine max
(L.) Merrill) or mung bean (e.g., Vigna radiata (L.) R. Wilcz.) by
the method as described above. The ACE inhibitory activity of the
tripeptides or tripeptide-rich compositions can be measured by, for
example, an in vitro test method and/or an in vivo test method as
will be described in Examples hereinafter.
[0017] When each peptide of the invention is prepared by chemical
synthesis, the synthesis can be carried out by any of the solid
phase method and the liquid phase method conventionally employed in
synthesizing a peptide. The peptide of the invention obtained by
the synthesis can be purified by a purification procedure commonly
employed, for example, reverse phase high-performance liquid
chromatography, chromatography with the use of an ion exchange
resin or a high-porous polymer resin, affinity chromatography,
etc.
[0018] The tripeptides thus obtained and the composition enriched
with the tripeptides have a strong activity of inhibiting ACE and
exhibit the strong ACE inhibitory effect when they are taken
orally. Therefore, they are useful as highly potent ACE inhibitors.
Moreover, they are easily absorbed via the gastrointestinal tract
and are relatively stable under heat. Due to these characteristics,
they are also applicable to foods, drinks and medicinal
preparations in various forms.
[0019] Accordingly, the present invention provides an edible
composition which comprises one or more of the above-described
tripeptides and which is expected to have an angiotensin converting
enzyme inhibitory effect, an angiotensin converting enzyme
inhibitor and a hypotensive agent containing one or more of the
above-described tripeptides.
[0020] In a case where one or more of the tripeptides of the
invention are used in foods, drinks, drugs, etc., use may be made
of a tripeptide sufficiently purified from the thermolysin
digestion product of the protein fraction of sesame, etc., or use
may be made of a chemically synthesized product. Alternatively,
since the tripeptides of the invention have a high stability and a
strong ACE inhibitory activity, the above-described partially
purified product or the thermolysin digestion product or the
partially purified product thereof may be used as such as a
tripeptide-rich composition; in such a case also, a sufficient ACE
inhibitory activity will be obtained, and hence, this is a
preferred embodiment of the invention.
[0021] The edible composition according to the present invention is
produced by adding one or more of the above-described tripeptides
in an amount of from 0.001 mg to 100 mg, preferably from 0.01 mg to
20 mg and still preferably from 0.1 mg to 10 mg in a single intake
dose. The tripeptides of the invention are in the form of a solid
or a powder which can be easily handled and are highly soluble in
water. Also, the tripeptides can be well absorbed via the
gastrointestinal tract. Therefore, the tripeptides may be added to
foods at any stage by any method without particular restriction.
That is to say, the tripeptides can be added in the form of a
powder, a solution, a suspension, etc. at the starting stage, the
intermediate stage or the final stage of a food production process
by using a method commonly employed in the field of food industry.
Temporary, intermittent, continuous or daily intake of the edible
composition containing the tripeptides of the invention makes it
possible to inhibit angiotensin converting enzyme and obtain, for
example, a hypotensive effect. The foods and drinks may be in the
form of, for example, a solid, a semifluid or a fluid. Examples of
solid foods include general foods and health foods in the forms of
biscuits, sheets, pills such as tablets and capsules, granules,
powders and so on. Examples of semifluid foods include products in
the forms of pastes, jellies, gels and so on. Examples of fluid
foods include general drink products and health drinks in the forms
of juices, cold drinks, tea drinks, tonic drinks and so on. Such
foods or drinks may be supplied in the form of a nutrition
supplemental drink or a seasoning to enable us to continuously take
the tripeptides of the invention, thereby suppressing the risk of
blood pressure elevation.
[0022] The medicinal composition of the invention contains the
tripeptides of the invention in an amount similar to the edible
composition as described above. The medicinal composition of the
invention can be temporarily administered to a hypertensive patient
to suppress the angiotensin converting enzyme in the body and
thereby to obtain a hypotensive effect. Alternatively, the
medicinal composition of the invention can be continuously
administered safely, since the active ingredient originates in a
natural material. As an example of the diseases which can be
treated and/or prevented by the medicinal composition of the
invention, hypertension can be mentioned. It is preferable that the
medicinal composition is in the form of an oral preparation such as
tablets, capsules, dusts, granules or syrups. Examples of
preparations for parenteral administration include aseptic
solutions to be administered intravenously, intraarterially,
subcutaneously, intramuscularly or intranasally. Such a solution
may be in the form of a dry solid which is to be dissolved before
using. An injection preparation can be produced by dissolving an
effective amount of the tripeptide in physiological saline and
treating under aseptic conditions as commonly employed in producing
injection preparations.
EXAMPLES
[0023] Now, the present invention will be described in greater
detail by reference to the following Examples.
Method of Measuring ACE Inhibitory Activity
[0024] In the present invention, the ACE inhibitory activity
(IC.sub.50) was measured in accordance with the following method.
[0025] Buffer: 0.1 M HEPES, 0.3 M NaCl, 0.01% Triton-X (pH 8.3).
[0026] Enzyme: ACE from rabbit lung (Sigma). [0027] Dissolved in
the above buffer and adjusted to a concentration of 1 mU/50 .mu.l.
[0028] Substrate: Bz-Gly-His-Leu.H.sub.2O (Peptide Institute Inc.).
8.95 mg of the substrate was dissolved in 1 ml of dimethyl
sulfoxide and further diluted 5-fold with water (final
concentration: 4 mM).
[0029] 5 .mu.l of a sample containing the peptide of the invention
was pipetted into a 96-well microplate. After adding 25 .mu.l of
the buffer and 10 .mu.l of the enzyme, the mixture was thoroughly
stirred and incubated at 37.degree. C. for 5 minutes. After adding
10 .mu.l of the substrate, the mixture was reacted at 37.degree. C.
for 30 minutes. Then the reaction was ceased by adding 40 .mu.l of
0.1 N NaOH. After adding 20 .mu.l of a methanol solution of 1%
o-phthalaldehyde and allowing to stand at room temperature for 10
minutes, 100 .mu.l of 0.1 N HCl was added and the resultant mixture
was incubated at 37.degree. C. for 30 minutes. Then the amount of
His-Leu formed by the hydrolysis by ACE was determined by exciting
(at 355 nm) the fluorescent substance formed by the reaction
between the amino group in the histidine residue and
o-phthalaldehyde and measuring the fluorescence wavelength at 460
nm. Then the percentage inhibition by the peptide of the invention
was determined in accordance with the following equation and the
ACE inhibitory activity (IC.sub.50) was calculated. Percentage
inhibition={1-(A-a)/(B-b)}.times.100 [0030] A: Measurement of
fluorescence when the sample was added. [0031] a: Measurement of
fluorescence when the sample was added and the buffer was added as
a substitute for the enzyme. [0032] B: Measurement of fluorescence
when distilled water was added as a substitute for the sample.
[0033] b: Measurement of fluorescence when distilled water was
added as a substitute for the sample and the buffer was added as a
substitute for the enzyme.
Example 1
Production and Purification of Peptide
[0034] 2 L of water was added to 100 g of defatted sesame and the
pH value of the resultant mixture was adjusted to 12.0 to 12.5 by
adding NaOH. After stirring at 55.degree. C. for 1 hour, the
mixture was filtered to give a protein extract. HCl was added to
the protein extract to adjust the pH value to 4.0. After
centrifuging, sesame protein (weight on dry basis: 19.8 g) was
obtained.
[0035] To 10 g of the obtained sesame protein, 300 ml of water was
added and the pH value of the mixture was adjusted to 7.5 with
NaOH. Then 10 mg of thermolysin (Nacalai Tesque, 7000 PU/mg) was
added thereto and the mixture was reacted under gentle stirring at
65.degree. C. for 6 hours. After the completion of the reaction,
HCl was added to the reaction mixture to adjust to pH 4.0 and the
thermolysin was inactivated by heating to 90.degree. C. for 10
minutes. After heating, the thus formed precipitate was removed by
centrifugation and the supernatant was filtered through paper
filter (Toyo, No. 2). The filtrate was freeze-dried to give 5.9 g
of a peptide powder.
[0036] 80 mg of this peptide powder was dissolved in 2 ml of 10%
ethanol solution and subjected to gel filtration column
chromatography. The conditions employed were as follows. [0037]
Column: Bio-Gel P-2 (15 mm ID.times.820 mm L, Bio-Rad). [0038]
Eluent: 10% ethanol. [0039] Flow rate: 0.15 ml/min. [0040]
Detection: UV 210 nm.
[0041] The eluate from the column was collected in fractions at
intervals of 15 minutes with the use of a fraction collector. The
ACE inhibitory activity of each fraction was measured in accordance
with the method described above. As a result, the major ACE
inhibitory activity under the above conditions was observed in
fractions 32 to 38. These fractions were combined and freeze-dried.
This procedure was repeated three times and thus 37.5 mg of
peptides was obtained in total.
[0042] Next, 37.5 mg of the ACE inhibitory active peptides obtained
by the Bio-Gel P-2 gel filtration column chromatography was
dissolved in 2 ml of purified water and subjected to
high-performance liquid chromatography with the use of an ODS
column to thereby fractionate the peptides. The conditions employed
were as follows. [0043] Column: Develosil ODS-10 (20 mm
ID.times.250 mm L, Nomura Chemical). [0044] Mobile phase: Buffer A:
5% CH.sub.3CN, 0.1% TFA. [0045] Buffer B: 40% CH.sub.3CN, 0.1% TFA.
[0046] Gradient: 0 to 20 min: 0% Buffer B [0047] 20 to 80 min: 0 to
100% Buffer B. [0048] Flow rate: 10 ml/min. [0049] Detection: UV
210 nm.
[0050] Under the above conditions, the eluate was collected in
fractions at intervals of 1 minute with the use of a fraction
collector. A 5 .mu.l portion of each fraction was pipetted into a
96-well microplate and evaporated to dryness under reduced
pressure. Next, the residue was dissolved in 5 .mu.l of purified
water to give a sample for measuring the ACE inhibitory activity.
Then the ACE inhibitory activity of each fraction was measured in
accordance with the method described above. As a result, fractions
39, 52 and 54 showed strong ACE inhibitory activities. The 3
fractions were freeze-dried and thus a small amount of peptides was
obtained from each fraction.
Purification of ACE Inhibitory Peptide in Fraction 39
[0051] The freeze-dried peptide of fraction 39 was dissolved in 200
.mu.l of purified water and subjected to high-performance liquid
chromatography with the use of a C30 column to thereby fractionate
the peptides. The conditions employed were as follows. [0052]
Column: Develosil C30-UG-5 (10 mm ID.times.250 mm L, Nomura
Chemical). [0053] Mobile phase: Buffer: 10% CH.sub.3CN, 0.1% TFA.
[0054] Flow rate: 4 ml/min. [0055] Detection: UV 210 nm.
[0056] Under the above conditions, the eluate was collected in
fractions at intervals of 15 seconds with the use of a fraction
collector. A 5 .mu.l portion of each fraction was pipetted into a
96-well microplate and evaporated to dryness under reduced
pressure. Next, the residue was dissolved in 5 .mu.l of purified
water to give a sample for measuring the ACE inhibitory activity.
Then the ACE inhibitory activity of each fraction was measured in
accordance with the method described above. As a result, fractions
44 and 45 showed strong ACE inhibitory activities. The 2 fractions
were freeze-dried separately and thus a small amount of peptide was
obtained from each fraction. Next, these fractions were subjected
to amino acid analysis and TOF MS/MS analysis. As a result, it was
found that the peptide of fractions 44 and 45 was Leu-Ser-Ala.
Purification of ACE Inhibitory Peptide in Fraction 52
[0057] The freeze-dried peptide of fraction 52 was dissolved in 200
.mu.l of purified water and subjected to high-performance liquid
chromatography with the use of a C30 column to thereby fractionate
the peptides. The conditions employed were as follows. [0058]
Column: Develosil C30-UG-5 (10 mm ID.times.250 mm L) [0059] Mobile
phase: Buffer: 14% CH.sub.3CN, 0.1% TFA. [0060] Flow rate: 4
ml/min. [0061] Detection: UV 210 nm.
[0062] Under the above conditions, the eluate was collected in
fractions at intervals of 15 seconds with the use of a fraction
collector. A 5 .mu.l portion of each fraction was pipetted into a
96-well microplate and evaporated to dryness under reduced
pressure. Next, the residue was dissolved in 5 .mu.l of purified
water to give a sample for measuring the ACE inhibitory activity.
Then the ACE inhibitory activity of each fraction was measured in
accordance with the method described above. As a result, fractions
89 and 90 and fractions 96 and 97 showed strong ACE inhibitory
activities. The 4 fractions were freeze-dried separately and thus a
small amount of peptide was obtained from each fraction. Next,
these fractions were subjected to amino acid analysis and TOF MS/MS
analysis. As a result, it was found that the peptide of fractions
89 and 90 was Ile-Val-Tyr, while the peptide of fractions 96 and 97
was Val-Ile-Tyr.
Purification of ACE Inhibitory Peptide in Fraction 54
[0063] The freeze-dried peptide of fraction 54 was dissolved in 200
.mu.l of purified water and subjected to high-performance liquid
chromatography with the use of a C30 column to thereby fractionate
the peptides. The conditions employed were as follows. [0064]
Column: Develosil C30-UG-5 (10 mm ID.times.250 mm L, Nomura
Chemical). [0065] Mobile phase: Buffer: 17% CH.sub.3CN, 0.1% TFA.
[0066] Flow rate: 4 ml/min. [0067] Detection: UV 210 nm.
[0068] Under the above conditions, the eluate was collected in
fractions at intervals of 15 seconds with the use of a fraction
collector. A 5 .mu.l portion of each fraction was pipetted into a
96-well microplate and evaporated to dryness under reduced
pressure. Next, the residue was dissolved in 5 .mu.l of purified
water to give a sample for measuring the ACE inhibitory activity.
Then the ACE inhibitory activity of each fraction was measured in
accordance with the method described above. As a result, fractions
69 to 73 showed strong ACE inhibitory activities. The 5 fractions
were separately freeze-dried and thus a small amount of peptide was
obtained from each fraction. Next, fractions 69, 70, 72 and 73
among them were subjected to amino acid analysis and TOF MS/MS
analysis. As a result, it was found that the peptide of each of
these fractions was Leu-Val-Tyr.
Example 2
Production of Peptides by Chemical Synthesis
[0069] Using an automatic peptide synthesizer (Model ABI 430)
manufactured by Applied Biosystems, a purposed protected peptide
resin was synthesized by starting with the C-terminus and extending
the peptide chain successively by the BOC method in accordance with
the program.
[0070] After the completion of the construction of the peptide on
the resin, the protected peptide resin was dried. The protected
peptide thus obtained was deprotected and the peptide was removed
from the resin support by treating it with anhydrous hydrogen
fluoride (HF/p-Cresol 8:2 v/v, 60 minutes). The crude peptide thus
obtained was extracted with 90% acetic acid and then freeze-dried
to give a powdery solid. The crude peptide thus obtained was
further purified by high-performance liquid chromatography with the
use of an ODS column and thus the purposed peptide was obtained.
[0071] Column: YMC-Pack ODS-2 (30 mm ID.times.250 mm L, YMC).
[0072] Mobile phase: Buffer A: 5% CH.sub.3CN, 0.1% TFA. [0073]
Buffer B: 40% CH.sub.3CN, 0.1% TFA. [0074] Gradient: 0 to 10 min:
0% Buffer B [0075] 10 to 90 min: 0 to 100% Buffer B. [0076] Flow
rate: 20 ml/min. [0077] Detection: UV 220 nm.
[0078] The purity of the peptide thus purified was examined by
high-performance liquid chromatography with the use of an ODS
column. [0079] Column: Zorbax 300SB-C18 (4.6 mm ID.times.150 mm L,
Agilent Technologies). [0080] Mobile phase: Buffer A: 1%
CH.sub.3CN, 0.1% TFA. [0081] Buffer: B: 60% CH.sub.3CN, 0.1% TFA.
[0082] Gradient: 0 to 25 min: 0 to 100% Buffer B [0083] Flow rate:
1 ml/min. [0084] Detection: UV 220 nm. Synthesis of Leu-Ser-Ala
[0085] Using Boc-Ala (BrZ) resin (0.5 mmol) as the starting amino
acid resin support, the peptide chain was extended with the use of
2 mM portions of amino acid derivatives Boc-Ser and Boc-Leu. Then
purified Leu-Ser-Ala was obtained by the purification method
described above in Example 2. The purity of the purified product
measured by the method described above in Example 2 was 99.0%.
Synthesis of Val-Ile-Tyr
[0086] Using Boc-Tyr (BrZ) resin (0.5 mmol) as the starting amino
acid resin support, a peptide chain was extended with the use of 2
mM portions of amino acid derivatives Boc-Ile and Boc-Val. Then
purified Val-Ile-Tyr was obtained by the purification method
described above in Example 2. The purity of the purified product
measured by the method described above in Example 2 was 98.8%.
Synthesis of Leu-Val-Tyr
[0087] Using Boc-Tyr (BrZ) resin (0.5 mmol) as the starting amino
acid resin support, a peptide chain was extended with the use of 2
mM portions of amino acid derivatives Boc-Val and Boc-Leu. Then
purified Leu-Val-Tyr was obtained by the purification method
described above in Example 2. The purity of the purified product
measured by the method described above in Example 2 was 99.2%.
Example 3
Measurement of ACE Inhibitory Activity of Peptide
[0088] The ACE inhibitory activities of the 3 peptides obtained in
Example 2 were measured in accordance with the method described
above and IC.sub.50 values were determined. Table 1 shows the
results. As a control, the ACE inhibitory activity of the sesame
peptide powder obtained in Example 1 was also measured and the
IC.sub.50 value thereof was determined. TABLE-US-00001 TABLE 1
Inhibitory activity (IC.sub.50) Peptide .mu.g/ml .mu.M Leu-Ser-Ala
2.4 8.4 Val-Ile-Tyr 1.6 4.2 Leu-Val-Tyr 0.84 2.1 Peptide powder --
50.3
Example 4
Hypotensive Effect of Peptide on Spontaneous Hypertensive Rat
[0089] SHR rats aged 17 to 22 weeks were fasted overnight. Then
each of the 3 peptides obtained in Example 2 was orally
administered in a dose of 1 mg/kg. To a control group, the same
amount of water was orally administered for comparison. Before, and
until 24 hours after the administration, changes in systolic blood
pressure were measured (BP-98A, SOFTRON). FIG. 1 shows the
results.
Example 5
[0090] Using the synthetic products of Example 2, a cereal tea
drink was produced from the following ingredients.
[0091] Composition: TABLE-US-00002 roasted barley 60 g hot water
2000 ml
[0092] Peptides of Example 2 TABLE-US-00003 Leu-Ser-Ala 19 mg
Val-Ile-Tyr 18 mg Leu-Val-Tyr 18 mg
Production Method:
[0093] Hot water was added to roasted barley and heated to
90.degree. C. for 5 minutes. After cooling to 40.degree. C., the
mixture was filtered. Then water was added to the extract to adjust
the volume to 2000 ml. Next, the above peptides were added and
dissolved by stirring to give a cereal tea drink.
Example 6
Isolation and Quantitation of Leu-Val-Tyr from Proteinase Treated
Plant Seeds
[0094] Rice and oat grains were respectively weighed at 25 g, which
were then ground to provide powders. 50 ml of hexane was added to
each flower and the solvent was removed through a filter paper
(Whattman, No. 1). The same hexane treatment was repeated 4 times
in total. Hexane was removed from the residue on the filter paper
to provide 18.8 g of a defatted rice powder and 15.9 g of a
defatted oat powder, respectively.
[0095] Each of the defatted flour was weighed at 10 g, suspended in
200 ml of 0.01 N NaOH and stirred at 55.degree. C. for 1 hour.
After cooling to room temperature, the suspension was filtered
through a filter paper (Whattman, No. 1). The filtrate was adjusted
to pH 4.0 by an addition of 0.1 N HCl. The precipitate thus formed
was collected by centrifugation, freeze-dried to yield 0.38 g and
0.57 g of crude protein powders of rice and oat, respectively.
[0096] The obtained powder was weighed at 0.2 g, suspended in 10 ml
of 0.1 mM CaCl.sub.2. The suspension was adjusted to pH 7.5, and
0.2 mg of thermolysin (7,000 PU/mg, Nacalai Tesque) was added
thereto to effect the enzyme reaction under gentle mixing at
65.degree. C. for 6 hours. After the reaction period, pH was
adjusted to 4.0 by 1 N HCl and the thermolysin was inactivated by
heating the mixture at 90.degree. C. for 10 minutes. The
precipitate formed by the heating was removed by centrifugation at
3,000 rpm for 30 minutes. The supernatant was freeze dried to
provide peptide powders of 28.6 mg from rice and 87.8 mg from
oat.
[0097] Isolation and quantitation of Leu-Val-Tyr, as one of the
peptides of the invention, from the rice and the oat peptide
powders obtained above was carried out as follows.
i) Pre-Treatment on PD-10 Column
[0098] Each of the peptide powders of rice and oat was weighed at
20 mg, dissolved in 0.1 N acetic acid to be 5 mg/ml, filtered
through a micro-filter (Millex-HV, pore size 0.45 .mu.m, filter
diameter 13 mm, Millipore Corporation) to remove insoluble
components. A 2.5 ml portion of the filtrate was introduced into a
PD-10 column (desalting column, Amersham Biosciences) equilibrated
with 0.1 N acetic acid. The column was washed with a further 3.5 ml
volume of 0.1 N acetic acid. Then the fraction eluted with an
additional 3.0 ml volume of 0.1 N acetic acid was collected,
evaporated to dryness, dissolved in 0.5 ml of water and then
freeze-dried.
ii) Gel Filtration HPLC with the Use of TSK-GEL G2000SWXL
[0099] The specimen prepared by the pre-treatment on the PD-10
column was dissolved in 250 .mu.l of 45% CH.sub.3CN, 0.1% TFA to be
centrifuged at 2,000 rpm for 5 minutes. The filtrate was filtered
through a micro-filter (Millex-HV, pore size 0.45 .mu.m, filter
diameter 13 mm, Millipore Corporation) to remove insoluble
components.
[0100] A 50 .mu.l portion of the filtrate was charged to a column
of TSK-GEL G2000SWXL (7.8.times.300 mm, Tosoh Corporation)
equilibrated with 45% CH.sub.3CN, 0.1% TFA, and HPLC was performed
with 45% CH.sub.3CN, 0.1% TFA (flow rate 0.7 ml/min., detection
wavelength 280 nm). The eluate of 1 minute between 30 seconds
before and after the retention time was collected, evaporated to
dryness, dissolved in 0.5 ml of water and freeze-dried. The
retention time of Leu-Val-Tyr was pre-determined by separately
subjecting synthetic Leu-Val-Tyr to HPLC under the same
conditions.
iii) Reverse HPLC on Develosil C30-UG-5 (Quantitation of
Leu-Val-Tyr)
[0101] Leu-Val-Tyr in the active peptide fractions from the gel
filtration was quantitatively analyzed by reverse HPLC on a
Develosil C30-UG-5 column (3.times.150 mm, Nomura Chemical Co.,
Ltd.). The fraction from the gel filtration HPLC on TSK-GEL
G2000SWXL was dissolved in 250 .mu.l of 5% CH.sub.3CN, 0.1% TFA,
centrifuged at 2,000 rpm for 5 minutes, and the supernatant was
filtered through a micro-filter (Millex-HV, pore size 0.45 .mu.m,
filter diameter 13 mm, Millipore Corporation) to remove insoluble
components. A 50 .mu.l portion of the filtrate was charged on a
Develosil C30-UG-5 column equilibrated with 5% CH.sub.3CN, 0.1% TFA
to perform a chromatography under the following conditions: [0102]
Elution solvent [0103] 0-5 min.: 5% CH.sub.3CN, 0.1% TFA [0104]
5-10 min.: 5-14% CH.sub.3CN, 0.1% TFA [0105] 10-35 min.: 14%
CH.sub.3CN, 0.1% TFA [0106] Flow rate: 0.4 ml [0107] Detection
wavelength: 280 nm
[0108] The peak of the each peptide of rice and oat in the
Develosil C30-UG-5 column chromatography, of which the retention
time corresponded to that of the authentic Leu-Val-Tyr of the
peptide, was collected. The fraction was subjected to a TOF MS
analysis and a TOF MS/MS analysis to confirm that the fraction was
Leu-Val-Tyr.
[0109] A calibration curve was prepared by charging different
amounts of the authentic Leu-Val-Tyr to the same Develosil C30-UG-5
column under the same conditions as described above and plotting
the peak areas against the charged amounts. Calibration Curve
Y=249197X-2150.6(R2=0.9991) [0110] Y: peak area, X: amount of
Leu-Val-Tyr (.mu.g)
[0111] The peak areas of the Leu-Val-Tyr fractions from the
Develosil C30-UG-5 chromatography of rice and oat were applied to
the calibration curve. As a result, the amounts of Leu-Val-Tyr in 1
mg of the peptide from rice and oat were determined to be 0.71
.mu.g and 1.05 .mu.g, respectively.
* * * * *